A wide spectrum of diseases with unmet medical need share some common pathogenesis that may be treatable, in principle, with protein kinase C (PKC) activators and Glycogen synthase kinase-3β (GSK-3β), inhibitors, respectively. Three such disease states, Alzheimer's disease (AD) (a neurological central nervous (CNS) disease), and Insulin resistance syndrome (IRS) and type-2 diabetes (T2D) (two metabolic diseases which are related to each other), are described briefly below. There is a close connection between IRS/T2D and AD (Sima and Li, Rev. Diabetic Stud. 2006, 3:161-168).
AD is a degenerative brain disorder characterized clinically by progressive loss of memory, by synaptic loss, by the presence of neuritic plaques consisting of β-amyloid (Aβ), by the presence of neurofibrillary tangles (NFT), and by loss of cholinergic neurons in the basal forebrain. Aβ are neurotoxic peptides. Tau (τ) are microtubule-associated proteins necessary for neurite outgrowth. Hyperphosphorylated tau proteins are in fact toxic and are the principal component of paired helical filaments (PHF) and NFTs. Insulin resistance induces chronic peripheral insulin elevations, reduces insulin activity, and reduces brain insulin levels. IRS and associated conditions such as T2D and hypertension are associated with age-related memory impairment and AD (Sima and Li, Rev Diabetic Stud 2006, 3:161-168).
A number of kinases are involved in AD pathology and IRS/T2D. Thus the amount of protein kinase C (PKC) is decreased in the brains of people suffering from AD, and this decrease has been shown to be correlated with neuropathological staging. This emphasizes the importance of this kinase as a major therapeutic target in AD (Kurumatani et al Brain Res 1998; 796:209-21).
Another kinase, GSK-3β, plays an important regulatory role in a multitude of cellular processes, ranging from cell membrane-to-nucleus signaling, gene transcription, translation, cytoskeletal organization to cell cycle progression and survival (Eldar-Finkelman, Trends Molec Med. 2002, 8:126-32; Bhat et al., Neurosignals 2002, 11:251-61; Balaram et al., Cell Mol Life Sci 2006, 63:1226-35). GSK-3β has been linked to most of the primary abnormalities associated with AD such as AD tau hyperphosphorylation, Aβ-induced neurotoxicity and presenilin-1 (PS-1) mutation pathogenic effects. Active GSK-3β triggers signal transduction events that participate in cell death, indicating that part of AD pathology could result from abnormal GSK-3β expression and activity. Furthermore, inactivation of GSK-3β has been correlated with decreased Aβ secretion (Sun et al., Neurosci. Lett. 2002, 321:61-4). Presently it is hypothesized that GSK-3β is the missing link between the β-amyloid and tau-pathology, placing GSK-3β as prominent player in the pathogenesis in AD [Takashima, J Alzheimers Dis 2006, 9 (3 Suppl), 309-17].
GSK-3β phosphorylates glycogen synthase and regulates the glucose metabolism pathway. Thus GSK-3β is a central negative regulator in the insulin signaling pathway, and it may have a role in insulin resistance (Gasparini et al. Trends Pharmacol Sci 2002:23:288-92; Janssens et al. Investig New Drugs 2006; 24: 263-80).
Thus inhibition of GSK-3β may mimic the action of certain hormones and growth factors, such as insulin, which use the GSK-3β pathway. This strategy may permit the bypassing of a defective receptor (e.g. the insulin receptor), or another faulty component of the signaling machinery, so that the biological signal will take effect even when some upstream players of the signaling cascade are at fault, such as in non-insulin-dependent type 2 diabetes [Tanabe et al, PLoS Biol. 2008 (2): e37; Wagman et al, Curr Pharm Design, 2004, 10:1105-1137].
Treatment strategies for the diseases mentioned above may include PKC activators and GSK-3β inhibitors. This can be achieved in principle either via indirect (GPCR-mediated) or direct modulation of these kinases. In case of direct activators of PKC or inhibitors of GSK3β, the quest is for highly potent and selective ligands. However, such therapeutic strategies would not be free of adverse effects as their target kinases are involved in a plethora of processes and downstream cascades. Thus direct targeting of these kinases for their function in one pathway (and linked disease) will alter their function in another pathway and potentially give rise to serious side effects (off-target side effects).
Therefore the ideal therapy for compounds that directly target these kinases should modulate selectively the discrete pathway(s) involved in the disease state. Such kinases can be modulated from outside the cell membrane via GPCRs. GPCRs convert signals received from outside the cell into biological processes inside the cell via signal transduction pathways. Such signal transduction pathways modulated by GPCRs are elegant systems by which cells and organisms can amplify subtle signals to generate robust responses. This downstream amplification process allows for clinical development of partial agonists that have a moderate binding potency and do not cause desensitization of the GPCR-mediated signaling following prolonged treatment in chronic disease states such as AD, IRS and T2D.
It is desirable for drug candidates for GPCR-modulation to have selectivity for the target GPCR subtype in order to prevent activation of other GPCR subtypes. A subclass of GPCRs are the muscarinic receptors (mAChR). Five genetically distinct human muscarinic receptors designated M1-M5 have been cloned (Buckley et al. Mol. Pharmacol. 1989; 35: 469-76; Hulme et al. Ann Rev Pharmacol Toxicol 1990; 30: 633-73). M1 mAChR, prevalent in the cortex, hippocampus and striatum, has an important role in cognitive processing and in particular in short-term memory, which is impaired in AD. M1 selective muscarinic agonists may serve as an anti-dementia drug treatment. The therapeutic potential of such compounds should, in principle, be less affected than the cholinesterase inhibitors (AChE-Is) by the extent of degeneration of presynaptic cholinergic terminals, and thus may represent a more rational treatment for AD than the FDA-approved AChE-Is (Review: Fisher, Neurotherapeutics, 5: 433-42, 2008). A number of bicyclic spiro-compounds, some reported to be M1-selective agonists, have been disclosed (U.S. Pat. Nos. 4,855,290, 4,981,858, 4,900,830, 4,876,260, 5,053,412, 5,407,938, 5,534,520, 5,852,029, 7,049,321, 5,221,675, 7,349,251).
A relation between three of the major hallmarks characteristic of AD has been reported: the CNS cholinergic hypofunction, formation of Aβ peptide amyloid plaques and tangles containing hyperphosphorylated tau proteins. In this context, vicious cycles link the cholinergic hypofunction in AD with Aβ peptide and tau phosphorylation. Stimulation of M1 mAChRs can increase cleavage of amyloid precursor protein (APP) in the middle of its β-amyloid region. This cleavage produces the secreted, neurotrophic and neuroprotective APPs (α-APPs), preventing the formation of Aβ peptide. M1 agonists may be of value in preventing Aβ formation by selectively promoting the α-secretase processing pathway in AD. Furthermore, stimulation of M1 mAChRs can decrease tau hyperphosphorylation (Review: Fisher, Neurotherapeutics 5:433-42, 2008). Thus some of the GPCR subtypes, and in particular the M1 mAChR, are involved in modulation of a multitude of functions, both in health and disease. PKC can be activated by several GPCRs including, but not limited to, M1 mAChR, metabotropic receptors and Wnt signaling (Farias et al., Neurobiol. Dis. 2004, 47:337-48; Mudher et al., J. Neurosci. 2001, 21:4987-95; Ballou et al., J. Biol. Chem. 2001, 44: 40910-916).
GPCRs in general can contain more than one site. In this context, the mAChR subtypes contain both orthosteric (primary binding site of the natural neurotransmitter, acetylcholine) and allosteric sites (may or may not alter the orthosteric site and the effects of acetylcholine).
Many of the pathological features of CNS and PNS diseases including AD and IRS/T2D, respectively, involve oxidative stress-related features. An oxidative stress in AD caused by Aβ can propagate a chain of events and vicious cycles leading to a blockade of some GPCR-induced signal transduction (best documented for M1 mAChR) and further accumulation of neurotoxic Aβ. Antioxidants can, in principle, prevent such vicious cycles (Fisher, Jap. J. Pharmacol. 2000, 84: 101-12; Kelly et al., Proc. Nat'l Acad. Sci. USA 1996 93:6753-58).
Oxidative stress can ultimately lead to both the onset and subsequent complications of T2D. Although antioxidant treatments can show benefits in animal models of diabetes, new and more powerful antioxidants are needed to demonstrate whether antioxidants can be effective in treating complications. Furthermore, it appears that oxidative stress is only one factor contributing to diabetic complications; thus, antioxidant treatment would likely be more effective if it were coupled with other treatments for diabetic complications. In particular, novel pathways that involve metabotropic receptor signaling (e.g. GPCR-mediated signaling), and GSK-3β, may be involved in diabetes and would need to be addressed in a comprehensive therapeutic strategy (Maiese et al, Curr Med. Chem. 2007 14:1729-38. Review).
Oxidative stress can lead to mitochondrial dysfunctions. Mitochondrial dysfunction exists in several neurodegenerative disorders (e g Alzheimer's Disease, Parkinson's Disease, Lewy Body Disease, Progressive Supranuclear Palsy, Amyotrophic Lateral Sclerosis, Frontotemporal Lobar Degeneration) and metabolic diseases (Type-2 Diabetes, insulin resistance) (review: Moreira et al, Antioxid Redox Signal, 9: 1621-1630, 2007; Reddy and Reddy, Curr Alzheimer Res 8:393-409, 2011; Sweedlow), Mitochondrial dysfunction can lead to cell death via apoptosis (programmed cell death) and this mitochondrial pathway is regulated by members of the Bcl-2 protein family (Antonsson, Cell Tissue Res, 306: 347-361, 2001; Reddy and Reddy, Curr Alzheimer Res 8:393-409, 2011). The intrinsic pathway of apoptosis is regulated by the Bcl-2 family of proteins which can be classified as proapoptotic or prosurvival (Youle et al, Nat Rev Mol Cell Biol 9, 47-59, 2008). Thus Bcl-2 protein itself is an apoptosis-suppressing factor, whereas another protein from the same family, Bax, is an apoptosis-promoting factor. The balance and location of these two types of proteins determines the fate of the cell. Bax is found in healthy cells in the cytosol, but upon apoptosis Bax undergoes conformational shift (e.g. dimerization; Gross et al, Genes Dev. 13:1899-19112010) and inserts in the outer mitochondrial membrane, being involved in mitochondrial outer membrane permeabilization, leading eventually to apoptosis. An elevated Bax/Bcl-2 ratio can be observed following insults leading to apoptosis, while anti-apoptotic agents can decrease this ratio and prevent apoptosis.